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Background: Despite spectacular advances in deciphering the molecular landscape of acute myeloid leukemia (AML), the clinical course of this disease has not improved dramatically for decades. Resistance to therapy and disease recurrence almost invariably occur, highlighting the need for a deeper understanding of these processes. Our general strategy takes into account decreased oxygen (O2) tension, a micro-environmental hallmark which remains understudied in the context of AML. While low O2 compartments such as bone marrow niches are well-recognized hosts of drug resistant AML cells, in vitro studies are still routinely performed in supra-physiologic ambient O2 concentration (21%), with potentially important consequences for clinical translatability. We hereby present an integrated molecular dissection of AML cells response to Cytarabine under O2-controlled conditions and exploit the information to develop novel synergistic/additive treatment combinations. Methods: M14, THP1, OCIAML3 and primary cells (PC) derived from AML patients of different risk groups were incubated in culture medium under 21% O2 or 1% O2 in the presence or absence of Cytarabine (Cy, 1-7.5 µM), Rosuvastatin (Ro, 10-200µM) and/or Pravastatin (Pra, 10-200µM). After 48h, proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Characterization of gene expression and expression levels of total and phosphorylated proteins were assessed using RNAseq and Reverse Phase Protein Arrays (RPPA), respectively. Gene Set Enrichment Analysis was performed following GSEA User Guide. Quantitative PCR reactions were performed using PowerUp SYBR Green PCR Master Mix (Applied Biosystems, USA) on 7900HT Real-Time PCR System. Intracellular cholesterol (ICC) levels were measured using WAKO Cholesterol assay kit. Results: Treatment with Cy under 1% O2 induced a specific gene signature of metabolic stress responses, including the perturbation of cholesterol homeostasis. Detailed analysis of the cholesterol pathway via RNAseq showed pathway induction by hypoxia itself and severe depletion by Cy. Focused RT-PCR assays on the cholesterol pathway components HMGCS1, MSMO1, LSS and SQLE demonstrated significant induction under 1% compared to 21% O2, and significant downregulation in response to Cy. In line with our RNAseq and PCR data, RPPA showed that critical cholesterol synthesis pathways, such as ACC1, are downregulated in response to Cy. Surprisingly, we observed that ICC levels significantly increased after 48h in response to treatment with Cy (p Summary: Treatment with Cytarabine induces a genetic signature of metabolic stress response, including the perturbation of the cholesterol biosynthesis pathway, in AML cells. Depletion of intracellular cholesterol levels is counterbalanced by upregulation of CD36 expression in Cytarabine-treated AML cells under 21% and 1% O2 conditions. Rosuvastatin exerts antileukemic activity via downregulation of intracellular cholesterol levels, induction of apoptosis and inhibition of AML cell growth. Rosuvastatin displays greater antileukemic effects than pravastatin. Rosuvastatin combined with Cytarabine acts synergistically against AML cells. Conclusions: Further investigation of Rosuvastatin as a component of anti-AML therapy is warranted. Disclosures No relevant conflicts of interest to declare.

In this issue of Blood, Kanakry et al analyzed Epstein-Barr virus (EBV) DNA expression in peripheral blood cellular compartments from 2146 patients with various malignancies and found that cell-free plasma EBV DNA was a better marker of EBV+ disease than EBV DNA from peripheral blood mononuclear cells (PBMCs).1

Circulating microRNAs (miRNAs) are potential biomarkers for cancer. We examined plasma levels of 2 miRNAs, let-7a and miR-16, in 50 patients with myelodysplastic syndrome (MDS) and 76 healthy persons using quantitative real-time PCR. Circulating levels of both miRNAs were similar among healthy controls but were significantly lower in MDS patients (P = .001 and P < .001, respectively). The distributions of these 2 miRNA levels were bimodal in MDS patients, and these levels were significantly associated with their progression-free survival and overall survival (both P < .001 for let-7a; P < .001 and P = .001 for miR-16). This association persisted even after patients were stratified according to the International Prognostic Scoring System. Multivariate analysis revealed that let-7a level was a strong independent predictor for overall survival in this patient cohort. These findings suggest that let-7a and miR-16 plasma levels can serve as noninvasive prognostic markers in MDS patients.

High-level leukemia cell expression of micro-RNA 155 (miR-155) is associated with more aggressive disease in patients with chronic lymphocytic leukemia (CLL), including those cases with a low-level expression of ζ-chain-associated protein of 70 kD. CLL with high-level miR-155 expressed lower levels of Src homology-2 domain-containing inositol 5-phosphatase 1 and were more responsive to B-cell receptor (BCR) ligation than CLL with low-level miR-155. Transfection with miR-155 enhanced responsiveness to BCR ligation, whereas transfection with a miR-155 inhibitor had the opposite effect. CLL in lymphoid tissue expressed higher levels of miR155HG than CLL in the blood of the same patient. Also, isolated CD5(bright)CXCR4(dim) cells, representing CLL that had been newly released from the microenvironment, expressed higher levels of miR-155 and were more responsive to BCR ligation than isolated CD5(dim)CXCR4(bright) cells of the same patient. Treatment of CLL or normal B cells with CD40-ligand or B-cell-activating factor upregulated miR-155 and enhanced sensitivity to BCR ligation, effects that could be blocked by inhibitors to miR-155. This study demonstrates that the sensitivity to BCR ligation can be enhanced by high-level expression of miR-155, which in turn can be induced by crosstalk within the tissue microenvironment, potentially contributing to its association with adverse clinical outcome in patients with CLL.

Noncoding RNAs play a pivotal role in the pathogenesis of chronic lymphocytic leukemia (CLL). We hypothesized that microRNAs (miRs) are involved in the transition from monoclonal B-cell lymphocytosis (MBL) to CLL and tested miR-15a/16-1 cluster, miR-21, and miR-155 expression in purified B cells of normal individuals, individuals with MBL, and patients with CLL. When we analyzed 224 samples from 2 independent training and validation cohorts, we found that miR-155 was overexpressed in B cells from individuals with MBL, and even more so in B cells from patients with CLL, when compared with B cells from normal individuals. Furthermore, we were able to identify miR-155 in circulating microvesicles from both individuals with MBL and patients with CLL. Next, to examine the prognostic role of miR-155, we measured its expression level in plasma samples collected before treatment initiation in 228 patients with CLL. We found significantly higher miR-155 expression levels in patients who failed to achieve a complete response compared with those who experienced complete response. Our findings support the use of cellular and plasma levels of miR-155 as biomarkers for the risk of progression in individuals with MBL, as well as to identify patients with CLL who may not respond well to therapy.

Long non-coding RNAs (lncRNAs) form the largest part of the mammalian non-coding transcriptome and control gene expression at various levels including chromatin modification, transcriptional and post-transcriptional processing. LncRNAs are implicated in initiation and progression of several cancers.Cancer-associated genomic regions are regions showing high frequency of cancer related abnormalities, such as loss of heterozygosity or amplifications. One such widely studied CAGR is the 8q24.21 genomic region. One SNP of particular importance present at this locus is rs6983267, with the G allele of the SNP conferring increased risk of colorectal, prostate, breast and bladder cancers. CCAT2 is a lncRNA that spans this highly conserved region. CCAT2 has been shown to play an important role in inducing chromosomal instability and supporting cell proliferation and cell cycle arrest. Despite advances in diagnosis of MDS patients, the underlying mechanisms that lead to spontaneous induction of MDS remains poorly understood. Here we attempted to elucidate the role of CCAT2 and its specific alleles (G/T) in regulation of cellular processes that drive spontaneous tumorigenesis using a genetically engineered mouse model. We generated transgenic mice for each CCAT2 allele using random integration approach in C57Bl6/N background, expressing CCAT2 in all tissues of mice. In this study, we identified that CCAT2 plays an important role in regulation of normal hematopoiesis. Constitutive in vivo overexpression of each CCAT2 transcript in the mice resulted in spontaneous induction of widespread pancytopenias. CCAT2(G/T) BM biopsies displayed severe myeloid or erythroid hyperplasia, and dysplastic megakaryocytic proliferation, along with enhanced proliferation and excessive apoptosis. Interestingly, we identified two distinct phenotypes in CCAT2(G/T) mice with equal prevalence of MDS or mixed MDS/MPN. This suggests that CCAT2 overexpression might affect regulation of hematopoietic stem cells, disturbing their self-renewal or maturation capacity, and subsequently resulting in BM failure. Percentage of HSPCs was significantly reduced in BM of MDS mice, with increased presence of immature erythroid blasts and granulocyte-macrophage progenitors suggesting a block in differentiation. HSPCs of CCAT2(G/T) mice also showed increased frequency of cytogenetic aberrations, including breaks and chromosomal fusions. However, these mice don't develop sAML, suggesting CCAT2 is critical in initiation of MDS. We further identified significantly higher CCAT2 expression in the MDS patients as compared to healthy volunteers. Patients with sAML had significantly lower expression of CCAT2 as compared to patients with only MDS. To determine the mechanism by which CCAT2 induces genomic instability and myelodysplasia, we screened for several genes that have been previously reported to induce myelodysplasia as potential targets of CCAT2. Interestingly, EZH2 was downregulated in the BMCs of CCAT2(G/T) mice compared to WT littermates. EZH2 downregulation was observed in both MDS only and MDS/MPD mice. In CCAT2(G/T) mice, EZH2 and H3K27Me3 reduction was observed in hematopoietic stem and progenitor cells (HSPCs) as well as lineage positive bulk cells,suggesting that CCAT2 might induce alteration in EZH2 levels in the HSC compartment. Interestingly, we identified miR-26a and miR-26b, that were already reported to target EZH2, were significantly overexpressed only in BM of CCAT2-G mice. These data suggests that CCAT2-G regulates EZH2 expression primarily through regulation of target miRNAs. On the other hand, we identified EZH2 to interact preferentially to CCAT2-T compared to WT or CCAT2-G transcript. These data confirmed that EZH2 preferentially binds to the CCAT2 in an allele-specific manner. In conclusion, deciphering the role of CCAT2 in spontaneously induced myelodysplasia and cytopenias will help us further characterize the poorly understood MDS/MPN phenotype. CCAT2 mice can serve as a robust model for studying initiation of de novo MDS/MPN that does not progress to secondary AML, and as a pre-clinical model for evaluation of new therapies for MDS. It has high translational potential as CCAT2 can be developed into a diagnostic and prognostic marker, as well as a novel intervention target for MDS therapy. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) is characterized by phenotypical and functional alterations of different host immune components. Lenalidomide is an immunomodulatory agent which exerts both a direct antitumor effect and a pleiotropic activity on the immune system. Single-agent lenalidomide is active in patients with relapsed and refractory CLL, and the addition of an anti-CD20 monoclonal antibody to lenalidomide has shown synergism in vitro, as well as improved clinical responses in vivo. We designed a phase II study with the aim of determining the efficacy and tolerability of combined ofatumumab and lenalidomide for the treatment of relapsed or refractory CLL. We also wanted to investigate whether immune system and tumor microenvironment characteristics, such as circulating T and NK cell number and function, and plasma levels of chemokines, cytokines, and angiogenic factors could be correlated with the response to treatment and could be modulated during the course of the therapy. Ofatumumab was administered at a dose of 300 mg on day 1, 1000 mg on days 8, 15, and 22 during course 1, 1000 mg on day 1 during courses 3-6, and once every other course on day 1 during courses 7-24 (28-day courses). Lenalidomide (10 mg daily) was started on day 9 and continued for as long as a clinical benefit was observed. Thirty-four patients were enrolled. The overall response rate was 71%. Eight patients (24%) achieved a complete remission (CR) or CR with incomplete recovery of blood counts, including 9% with minimal residual disease-negative CR. The median progression-free survival was 16 months, and the estimated 5-year survival was 53%. The most common treatment-related toxicity was neutropenia (grade >2 in 18% of the 574 patient-courses). The most frequent infectious complications were pneumonia and neutropenic fever (24% and 9% of patients, respectively). For the correlative studies, peripheral blood mononuclear cells and plasma samples were collected at baseline and after 3, 6, 9, and 12 months of therapy. Lymphocyte subsets were analyzed by flow cytometry. T and NK cell function was evaluated on 9 patients based on the expression of CD107a, IFNγ, TNFα, and IL-2 after the exposure to CD3/CD28 magnetic beads and K562 cell line, respectively. Chemokines, cytokines, and angiogenic factors were quantified through a multiplex bead-based immunoassay. Thirty-four age-matched healthy donors (HD) were used as controls. Treatment with ofatumumab and lenalidomide decreased the number of circulating T and NK cells. The greatest decrease was observed after 3 months of therapy as compared to baseline (p≤0.02 for all comparisons). We found that the baseline number of CD4+ T cells and CD57+ CD56+ NK cells was higher in CR patients as compared to non responder (NR) patients (median absolute number 1595/μl vs. 715/μl, p=0.04, and 358/μl vs. 70/μl, p=0.02). We also compared baseline T and NK cell populations of CR patients to HD, and found that the former had significantly higher numbers of CD16+ CD56+ and CD57+ CD56+ NK cells (median 356/μl vs. 80/μl, p=0.03, and 358/μl vs. 29/μl, p=0.0008). Interestingly, baseline numbers of CD16+ CD56+ and CD57+ CD56+ NK cells were comparable between NR patients and HD. We also observed that patients who achieved CR had significantly better T and NK cell function at baseline, whereas T and NK cell function was impaired in patients who only had partial or no response. Plasma levels of bFGF, VEGF and IFN-γ were significantly decreased at month 3 compared to baseline in all patients (p≤0.02 for all comparisons). Finally, significantly lower levels of circulating CCL2, CCL3, CCL4, IFNα, and IL-12p70 were detected during treatment in patients who achieved CR, compared to NR (p In conclusion, the combination of ofatumumab and lenalidomide is a well tolerated regimen that induces durable responses in the majority of patients with relapsed CLL, and sustained MRD-negative CR in some. A higher number and preserved functional activity of T and NK cells were found in responding patients at baseline, as compared to NR, underscoring the importance of a competent immune system in supporting the effectiveness of this type of treatment, and suggesting a need to further investigate these immune features as markers for prediction of response to therapy. Disclosures Faderl: Celgene Corp.: Other: Advisory Board. Wierda:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding. Burger:Pharmacyclics LLC, an AbbVie Company: Research Funding. Keating:Glaxo-Smith-Kline Inc.: Other: Advisory board; Celgene Corp.: Consultancy.

Aberrant expression of microRNAs (miRNAs) has been associated with clinical outcome in patients with chronic lymphocytic leukemia (CLL). To identify a powerful and easily assessable miRNA bio-marker of prognosis and survival, we performed quantitative reverse-transcription polymerase chain reaction (qRT-PCR) profiling in 104 CLL patients with a well-defined chromosome 17p status, and we validated our findings with miRNA microarray data from an independent cohort of 80 patients. We found that miR-15a, miR-21, miR-34a, miR-155, and miR-181b were differentially expressed between CLLs with chromosome 17p deletion and CLLs with normal 17p and normal karyotype, and that miR-181b was down-regulated in therapy-refractory cases. miR-21 expression levels were significantly higher in patients with poor prognosis and predicted overall survival (OS), and miR-181b expression levels significantly predicted treatment-free survival. We developed a 21FK score (miR-21 qRT-PCR, fluorescence in situ hybridization, Karyotype) to stratify patients according to OS and found that patients with a low score had a significantly longer OS time. When we evaluated the relative power of the 21FK score with the most used prognostic factors, the score was the most significant in both CLL cohorts. We conclude that the 21FK score represents a useful tool for distinguishing between good-prognosis and poor-prognosis CLL patients.

Resistance to chemotherapy is an unsolved medical problem and no clear solutions have been identified despite decades of research. In this issue of Blood , Zenz and colleagues identify miR-34a as a component of the chemotherapy resistance network in CLL. This is a remarkable finding with possible

The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1α under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1α can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL.

High BAALC expression levels are associated with poor outcome in cytogenetically normal acute myeloid leukemia (CN-AML) patients. Recently, miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML patients. Gene-expression profiling and miRNA-expression profiling were performed using microarrays. High miR-3151 expression was associated with shorter disease-free and overall survival, whereas high BAALC expression predicted failure of complete remission and shorter overall survival. Patients exhibiting high expression of both miR-3151 and BAALC had worse outcome than patients expressing low levels of either gene or both genes. In gene-expression profiling, high miR-3151 expressers showed down-regulation of genes involved in transcriptional regulation, posttranslational modification, and cancer pathways. Two genes, FBXL20 and USP40, were validated as direct miR-3151 targets. The results of the present study show that high expression of miR-3151 is an independent prognosticator for poor outcome in CN-AML and affects different outcome end points than its host gene, BAALC. The combination of both markers identified a patient subset with the poorest outcome. This interplay between an intronic miR and its host may have important biologic implications.

In this issue of Blood , Palamarchuk and colleagues present interesting evidence that deletions in chromosome 13q result in a loss of expression of both the protein coding gene, dleu7 as well as the noncoding RNA cluster miR-15a-16-1 . dleu7 and miR15a-16-1 may both have a role in the pathogenesis of CLL.

In this issue of Blood, Stamatopoulos and colleagues present evidence that down-regulation of miR-29c and miR-223 in malignant B cells is an adverse prognostic indicator in CLL. The findings in this paper are remarkable in that they help refine disease stratification and prognosis.

Abstract 2885 MicroRNAs (miRs) are involved in the initiation, progression and dissemination of CLL cells (Calin GA, Croce CM. Blood 114:4761, 2009). Recent studies showed that high levels of miR-155, previously shown to regulate hematopoietic cell development, are expressed in CLL cells. Because transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation, it is thought that miR-155 plays a role in the pathogenesis of CLL (Calin GA et al. N Engl J Med 353:1793, 2005). STAT3 is constitutively activated in CLL and induces the transcription of several STAT3-regulated genes. A recent study demonstrated that STAT3 activates miR-21 and miR-181b-1 (Iliopolus D. et al. Mol Cell 39:493, 2010). Therefor we wondered whether STAT3 enhances the expression of miR-155 in CLL cells. Because a sequence analysis revealed that the promoter of miRNA-155 harbors γ-interferon activation sequence-like elements typically activated by STAT3, we sought to determine whether STAT3 directly activates miR-155 expression. We generated truncated constructs of the miR-155 promoter, co-transfected them into MM1 cells together with STAT3 small interfering (si) RNA (siRNA), and assessed their luciferase activity. The luciferase activity data suggested that of the two putative STAT3 binding sites only one site is involved in STAT3 induced transcription because STATR3-siRNA reduced the activity of miRNA-155 promoter of constructs that harbor this site. To confirm these data we performed an electrophoretic mobility shift assay (EMSA) and chromatin immune-precipitation (ChIP). The EMSA confirmed that STAT3 bound the miR-155 promoter in fresh CLL cells, and ChIP confirmed that STAT3 bound one putative STAT3-binding site in the miR-155 promoter but not to the other, as demonstrated by the luciferase assay; STAT3 co-immuno-precipitated only one putative STAT3 binding region of miR-155 promoter and other STAT3-regulated genes. Finally, STAT3-small hairpin RNA (shRNA) downregulated miR-155 and other STAT3-regulated genes, suggesting that constitutively activated STAT3, binds miR-155 promoter and induces miR-155 transcription in CLL cells. Disclosures: Keating: Celgene Corporation: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Xcenda: Consultancy, Speakers Bureau.

Abstract 2886 Non-coding RNAs regulate the expression of more than 30% of protein-coding genes both at a post-transcriptional and translational level. Although approximately 1000 microRNAs (miRs) have been identified in the human genome, little is known about the mechanisms that regulate miR expression. STAT3 regulates the transcription of miR-21, and miR181b-1, binds to their promoter and induce neoplastic cell transformation (Iliopoulos, Jaeger et al. 2010). Because STAT3 is constitutively activated in CLL cells (Hazan-Halevy, Harris et al. 2010) we sought to investigate how STAT3 affects non-coding RNA gene expression in CLL cells. We transfected peripheral blood CLL cells from 3 different patients with STAT3-shRNA and assessed non-coding RNA levels using a non-coding RNA array containing 2277 human miR probes, 960 from ultra-conserved genes and 3540 of long non-coding RNAs. When compared to transfection control, 152 probes from 78 non-coding RNA genes were differentially expressed (134 down-regulated and 18 up-regulated), suggesting that STAT3 affects the non-coding RNA network in CLL cells. Supervised clustering analysis was used to select genes for validation. By using quantitative RT-PCR we validated our gene array analysis. Similar to the data obtained by the non-coding RNA array, we found that transfection of CLL cells with STAT3- down-regulated the levels of miR-21, miR-155, and miR-320b. Binding site prediction programs and ChIP-seq data embedded in the UCSC genome browser determined that in 5 of 7 genes, down-regulated by STAT3-shRNA transfection, were either putative or experimentally confirmed STAT3-binding sites, indication that STAT3 directly regulates the transcription of those miRs. It has been shown that the interaction between miRs and single stranded RNA is dependent on base pairing in a seed region at positions 2 to 8. High levels of 4.8kb single stranded STAT3 RNA transcripts, present in CLL cells, provide a substrate for such paring. Therefore, we assumed that STAT3 functions as a “RNA sponge” soaking up miRs and altering their effective levels and function. To test this hypothesis we used the pattern-based RNA22 algorithm and identified potential miR targets. We than calculated the energy that would be released if the corresponding RNA/RNA complexes are saturated. We found that the energy released from binding of miRs to STAT3 sequences would be higher than energy released from binding to a random sequence with same length and base content suggesting that STAT3 “sponges out” miRs in a sequence specific manner. Thus, CLL cells are characterized by an ongoing interaction between STAT3-mediated transcriptional regulation of non-coding RNA and miR-mediated translational regulation of coding genes. Disclosures: No relevant conflicts of interest to declare.

Abstract 3916 Introduction: Multiple myeloma is clinically and biologically heterogeneous. Certain translocations and chromosomal losses (t{14;16}, t{14;20}, del 17p) and gene expression profiles define high-risk disease. Recently, several groups found microRNAs (miRs) dysregulated in multiple myeloma. A profile composed of 28 miRs was found to define high-risk disease. Among the dysregulated miRs, miR15a, miR16 were down-regulated, miR19b, miR20a, miR181b were increased according to most publications. MiR-21 was generally upregulated in high-risk disease and inducible by interleukin-6. We hypothesized that commonly administered treatments for multiple myeloma would alter the expression pattern of these miRs. Materials and Methods: For these in-vitro experiments, 4 established cell lines were used: RPMI8226, OPM-2 (t4;14), Kas-6 (IL-6-dependent) and MM1-S (t14;16). The cells were treated with ionizing radiation (3- 6 Gy), lenalidomide (10 μM), doxorubicin (50 ng/ml), bortezomib (2- 50 nM), SAHA (1–3x 10−6 M), pegylated interferon α (3–300 ng/ml) and nutlin-3 (10 μM) between 2 and 48 hours. RNA was extracted and quantitative real-time RT-PCR was performed for miR-15a, miR-16, miR19b, miR-20a, miR21, miR-181b and a control gene (U6). The expression was calculated and compared by the ΔΔ CT method. Results: Ionizing radiation increased miR15a in 1/2 cell lines at early time points, increased miR-19b at early time points in 2/2 cell lines (decreased later) and increased MiR20a in 2/2 cell lines at early time points. Lenalidomide induced miR15a in 2/4 cell lines, miR19b in 3/ 4 cell lines and miR-20a in 3/ 4 cell lines. Doxorubicin increased miR-16 in 2/3 cases and miR-20a in 2/3 cases (in 1 cell line decreased). Bortezomib overall induced few changes in miR-expression. SAHA induced miR-15a in 2/3 cell lines and decreased miR-16 in 1/3. MiR-19a was decreased with SAHA in 2/4 and increased in 1/4 cell lines. MiR-20 decreased in 1/4 and increased in 1/4. MiR-21 decreased in 1 and increased in 1/4 SAHA-treated cell lines. MiR-181b increased in 2/4 cell lines. Pegylated interferon decreased MiR-15a in 3/4 cell lines, decreased miR-16a in 3/4 cell lines, increased miR19b in 2/4 cell lines. MiR-20a was increased in 2/4 and decreased in 1/4 cell lines. MiR-181b was decreased in 2/ 4 cell lines. Nutlin-3 increased miR16 in 1/3 cell lines, increased miR-20a in 2/4 cell lines, increased miR-181b in 2/4, decreased miR-181b in 1/4 cell lines. Most changes observed are in the range of −50 – + 200%. Conclusions: Many miRs are induced at early time points under non-cytotoxic conditions. The variability observed in these experiments may be due to the genetic heterogeneity of the cell lines. Interferon mostly down-modulates the expression of the miRs studied. Previous experiments, for example using endothelial cells also showed an induction of certain miRs after cytotoxic or cytostatic treatments. This can be explained as a stress response or protective mechanism enhancing tumor cell survival. However, the functional relevance of our data was not investigated. The downregulation of miRs following interferon treatment is surprising and would argue for a combination of interferon with cytostatic treatments. If confirmed using CD138 selected samples from patients with multiple myeloma, our data may be used to develop a treatment profile which ultimately might prognosticate treatment response. Our results are also relevant for future miR-based treatments for multiple myeloma. Disclosures: Orlowski: Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 609 Introduction: Myelodysplastic syndromes (MDS) represent a very complex group of myeloid leukemias with diverse genetic and epigenetic characteristics. We have identified JMJD3, a histone demethylase, aberrantly upregulated in bone marrow CD34+ hematopoietic progenitor cells of MDS (Blood 2009). JMJD3 is a jmjc domain containing histone demethylase that removes methyl groups from Lys-27 of histone H3, and is also known to positively regulate the methylation on Lys-4 of histone H3. This results in activation of upstream regulators of NF-kB signaling, suggesting that JMJD3 plays a crucial role in the pathogenesis of MDS. Therefore, insight on the regulation of JMJD3 expression in MDS is of importance. Aberrant expression of miRNAs, including loss of expression, which leads to up-regulation of the expression of its target genes, has been identified in various types of malignancies including MDS. We hypothesized that alteration of regulatory miRNAs could be involved in the upregulation of JMJD3 in MDS CD34+ cells. Identification of JMJD3 regulatory miRNAs may help further dissect the molecular mechanisms underlying the pathogenesis of MDS. Methods and Results: To identify regulatory miRNAs targeting JMJD3, we performed an analysis using microRNA Genomics Resource (miRGen) and identified 40 candidate miRNAs that potentially interact with the 3′ untranslate region (3′-UTR) of JMJD3 RNA. We then characterized the interactions of these candidate miRNAs to JMJD3 by using a luciferase assay system by cloning the 3′-UTR region of JMJD3 gene into a luciferase reporter construct. By co-transfecting each of the 27 available miRNAs from the 40 candidates together with the luciferase reporter construct into human megakaryocytic blast cell line Meg-O1, we identified 7 mirRNAs (has-mir -29a, -29b, -29c, -99b, -101, -377, and -767-5p) that negatively regulate JMJD3 3′UTR associated luciferase activity. We then examined the expression for these 7 key potential regulatory miRNAs of JMJD3 in a cohort of 36 patient bone marrow CD34+ cells by microRNA Taqman probe based Q-PCR analysis. The median age of these patients was 67 years (32 to 85); 42% had low or INT-1 disease; 21% were diploid and 28% had an alteration of chromosome 5 or 7. Analysis showed that 16 of the 36 MDS CD34+ cell samples examined showed lost expression of mirRNA hsa-mir767-5p, and 7 had down-regulated expression of the same microRNA. For these 23 MDS samples with repressed mir-767-5p expression, 11 of them (~48%) had upregulated expression of JMJD3, as demonstrated by Q-PCR analysis Consistently, in miRNA transfected Meg-O1 cells, Q-PCR analysis indicates that over-expression of mir767-5p is accompanied with decrease of JMJD3 RNA by 68% based on Q-PCR analysis using 3 different probes for JMJD3. Of interest, 4 of the 5 patients with monosomy of chromosome 7 had downregulation of mir767-5p. Conclusion and Future Direction: These results support the hypothesis that mir767-5p is a key regulatory miRNA targeting JMJD3 in hematopoietic progenitor cells. Since one important mechanism for miRNA on target gene expression is through inhibiting the translation from mRNA to protein, we are now developing an antibody against endogenous JMJD3, with which we will be able to further correlate the level of mir767-5p and the expression of JMJD3 protein in both cell lines and primary MDS samples. Furthermore, correlation between alteration of mir767-5p with clinical, cytogenetic and molecular features in MDS patients will be characterized in a larger cohort of patients. These will further determine the role of mir767-5p for the regulation of JMJD3 and its role during the pathogenesis of MDS. By pursuing the studies described above, a potential link between two key epigenetic regulatory components, histone methylation regulator (JMJD3) and microRNAs, involved in pathogenesis of MDS, will be established. Disclosures: No relevant conflicts of interest to declare.

Abstract 1701 Introduction: MicroRNA (miRNAs) are noncoding RNAs of 19 to 25 nucleotides in length that regulate gene expression by inducing translational inhibition and cleavage of their target mRNAs through base pairing to partially or fully complementary sites. Altered miRNA expression has been associated to various types of cancers, including leukemia. Recently it has been reported that pretreatment expression levels of miR-29b are associated with clinical response in pts with AML treated with a 10-day schedule of decitabine (PNAS 2010; 107:7473-78). These findings are significant as detection of miR-29b, if validated, could be used to select therapy in pts with AML. Methods: To follow on these observations, we have analyzed miR-29b and miR-101 expression levels in a cohort of patients with AML or high-risk myelodysplastic syndrome (MDS) previously treated in I/II study of the combination of 5-azacitidine (5-AZA), valproic acid (VPA) and ATRA (Blood 2007; 110:2302-08). We analyzed 45 pts, including 28 previously untreated patients. The characteristics of these pts have been previously reported (above): 8 (29%) pts achieved a complete remission and 1 (4%) partial remission. Total cellular RNA (extracted from unselected peripheral blood mononuclear cells collected before the first cycle of treatment) was used for reverse transcription reactions using TaqMan microRNA reverse transcription kit (Applied Biosystems). For real - time PCR, miR-29b and miR-101 TaqMan - microRNA assays were purchased from Applied Biosystems and analyzed with TaqMan Universal PCR Master Mix (Applied Biosystems) using an Applied Biosystems Prism 7500 Sequencing detection system. The U6 small nuclear RNA was used as internal control. Results: Expression levels of miR-29b and miR-101 from 45 patients were compared to 6 healthy donors. Expression levels of miR-29b was down-regulated in patients (P=0.001) (Fig. A). There were no difference in terms of miR-101 expression between patients and health donors. We then compared the miR-29b and miR-101 expression levels between patients who responded to the combination therapy versus non-responders: no differences were observed for both miR-29b (P=0.56) and miR-101 expression (Fig. B). Conclusion: our results fail to detect a relationship between miR-29b levels and response to 5-azacitidine based therapy. This could be explained by the fact that: 1) in our study, we used 5-azacitidine in combination versus decitabine; 2) source of RNA (peripheral blood versus bone marrow); And 3) relatively small sample size. Despite these limitations, these results indicate that larger studies need to be performed to confirm the role of miRNAs in response to hypomethylating agent based therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 855 MicroRNAs (miRs) and heterogeneous ribonucleoproteins (hnRNPs) are post-transcriptional gene regulators that bind mRNA in a sequence-specific manner. We have reported that a) hnRNP-E2 suppresses CEBPA mRNA translation and inhibits myeloid maturation of bone marrow (BM) progenitors from chronic myelogenous leukemia patients in myeloid blast crisis (CML-BCCD34+; Perrotti et al, Nat Genet 2002); and b) miR-328 expression is lost in myeloid CML-BCCD34+ progenitors (n=6) and its restored expression at physiological levels rescues granulocytic differentiation and impairs clonogenic potential of primary BCR/ABL+ blasts (Eiring et al, ASH 2007). Here we show by Northern blot, real-time PCR, and microarray analyses that miR-328 levels increase during granulocytic differentiation of normal human CD34+ and mouse Lin− BM progenitors, but not during differentiation towards erythroid, megakaryocytic or monocytic lineages. BCR/ABL uses the same MAPKERK1/2-hnRNP-E2 signaling pathway to suppress both C/EBPα and miR-328, as pharmacologic or shRNA-mediated inhibition of these molecules restored miR-328 expression in BCR/ABL+ cells. In fact, two functional C/EBPα binding sites are present in the miR-328 promoter region and C/EBPα interacts in vivo with these regulatory elements to enhance miR-328 transcription. Importantly, we also show that restored maturation of BCR/ABL+ blasts requires direct interaction of hnRNP-E2 with the C-rich regions of miR-328. Indeed, RNA-immunoprecipitation (RIP) assays demonstrated that miR-328 directly binds to hnRNP-E2 independent of the RNA-induced silencing complex (RISC). Furthermore, ectopic miR-328, but not miR-181b, resulted in decreased in vivo binding of hnRNP-E2 to the uORF/spacer region of CEBPA mRNA, thereby releasing CEBPA from hnRNP-E2 translation inhibition and rescuing C/EBPa-driven neutrophil maturation (decoy activity). Differentiation of miR-328-expressing CML-BCCD34+ blasts (88.8±2.4% post-mitotic cells) correlated with induction of C/EBPa protein expression, whereas CEBPA mRNA and hnRNP E2 protein levels remained unchanged. The existence of a direct miR-328/hnRNP-E2/CEBPA interplay was formally demonstrated in vitro using RRL-directed translation assays and in vivo using the 6.15 clone of 32D-BCR/ABL cells that do not express endogenous CEBPA mRNA and require ectopic C/EBPα (wt-uORF-CEBPA) for differentiation. Addition of miR-328, but not miR-330, to hnRNP-E2-containing RRL reactions increased newly synthesized 35S-C/EBPa levels by >100%. Likewise, forced miR-328 expression in vivo resulted in decreased hnRNP-E2 binding to CEBPA mRNA, induction of C/EBPa protein but not mRNA and rescued granulocytic differentiation of 6.15-wt-uORF-CEBPA but not vector-transduced 6.15 cells. While hnRNP-E2 was not found in complex with basic RISC components (Dicer, TRBP2 and Ago2), RIP assays detected miR-328 associated to Dicer and Ago2 in miR-328-expressing cells, suggesting that it also acts through canonical RISC-dependent base-pairing with mRNA targets. Indeed, we identified the BCR/ABL-regulated PIM1 serine-threonine kinase as a bona fide miR-328 target in BCR/ABL+ cells. Ectopic miR-328 suppressed PIM1 protein but not mRNA levels, and this effect required integrity of the miR-328 binding site present in the PIM1 3'UTR. Forced expression of a wild-type but not kinase-deficient PIM1 lacking the 3'UTR into miR-328-expressing cells fully rescued BCR/ABL clonogenicity, suggesting that miR-328-induced PIM1 suppression accounts for reduced survival of miR-328-infected BCR/ABL+ blasts. To show that miR-328 acts on PIM1 in a RISC-dependent manner, we mutated the miR-328 seed sequence (miR-328-Mut) while retaining its C-rich character. Similar to wild-type miR-328, miR-328-Mut efficiently interacted with hnRNP-E2, restored C/EBPa protein expression and rescued granulocytic differentiation, but was unable to silence PIM1 in 32D-BCR/ABL cells, indicating that the C-rich character of miR-328 is essential for its decoy activity, while its seed sequence integrity is necessary for RISC-dependent pairing to mRNA targets. Thus, the discovery of dual activities for miR-328 not only adds a new layer of complexity to the mechanisms regulating CML disease progression, but also highlights the ability of miRNAs to alter mRNA metabolism by acting as molecular decoys for RNA-binding proteins. Disclosures: Cortes: Novartis: Research Funding.

Background: B-Chronic Lymphocytic Leukemia (CLL) is predominantly characterized as a clonal B-cell disorder where a minor fraction of CLL B cells proliferate, but the majority of these cells are non-cycling. Importantly, CLL B cells also exhibit apoptosis resistance that results from a complex set of redundant mechanisms. This latter feature presents a significant problem in relation to disease progression and developing effective therapies in CLL. Previously, we and others have reported that leukemic CLL cells spontaneously secrete pro-angiogenic vascular endothelial growth factor (VEGF) that can significantly enhance CLL leukemic B cell apoptosis resistance via unknown mechanisms. In addition, the molecular mechanism of the constitutive upregulation of VEGF in CLL B cells is unknown. Methods and Results: Here, we report that CLL B cells express a high level of HIF-1α protein under normoxic conditions as compared to that of normal B cells in Western blot analysis using a specific antibody. Accumulation of HIF-1α in CLL B cells resulted in upregulation of its downstream target VEGF. To comprehend this abnormal overexpression of HIF-1α, we have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation by Western blot analysis and found it to be either absent or notably low level in CLL B cells as compared to that of normal B cells. Furthermore, we have shown by in vitro reporter gene assays that miR-92a, a microRNA known to be overexpressed in CLL B cells, can target the VHL 3′-untranslated region (UTR) and is likely a reason for the depressed levels of pVHL in CLL B cells. To validate our hypothesis, we transiently transfected the human embryonic kidney cell line (293T) with miR-92a and observed a dose-dependent repression of pVHL level. Post-transcriptional regulation of the VHL gene was further confirmed when we observed a subtle but definite increase of the pVHL levels monitored by immunoblot following nucleofection of the antisense oligo targeted to miR-92a into primary CLL B cells. To examine whether HIF-1α is functionally active in CLL B cells, we performed co-immunoprecipitation experiments and found that HIF-1α forms an active complex by physical association with the transcriptional co-activator p300 and phospho-STAT3 in CLL B cells. The latter molecule is known to be constitutively expressed in CLL B cells but its function has been unknown. Subsequently, we have found via chromatin immunoprecipitation analyses in CLL B cells that HIF-1α and STAT3 are bound to the VEGF promoter and also recruit RNA polymerase II, further substantiating that this complex likely functions in the activation of VEGF transcription. Summary: Overall, our data show that CLL B cells express constitutively elevated HIF-1α levels in normoxia due, at least in part, to miR-92a regulated diminished pVHL expression. This is a unique previously undescribed mechanism for enhanced HIF-1α levels in human malignancy. We believe that Increased VEGF expression is associated with the combined effects of HIF-1α, p300 and constitutively active STAT3 as a functional complex and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. These findings now provide additional strategy points for interrupting the VEGF autocrine pathway in CLL B cells.

UCRs are a subset of conserved sequences (100% between orthologous regions of human, rat and mouse genomes) located in both intra- and intergenic regions. There are 481 UCRs described and more than 50% of all the UCRs (256/481) has been classified as non exonic (with no evidence of coding protein), while the other 47% have been designated either exonic (overlap mRNAs of known genes), or possible exonic (with no clear evidence of overlap with protein coding genes) (Bejerano et al, Science 2004). Recently, our group reported that a subset of transcribed UCRs is aberrantly expressed in leukemia (Calin et al, Cancer Cell 2007). Because of the high degree of conservation, the UCRs are likely to be functional and may be involved in the phylogeny of mammals. Since hematopoiesis is regulated by signaling pathways and transcription factors that are highly conserved throughout phylogeny, we hypothesize that UCRs may be differentially expressed in hematopoietic tissues and may play a role in the regulation of this process. In this study we analyzed the UCRs expression in normal hematopoiesis by performing microarray analysis of hematopoietic precursors (HPCs) obtained by culturing human bone marrow (non-mobilized) CD34+ selected cells with different cytokine combinations for 2 weeks to stimulate differentiation to the erythrocyte (E), megakaryocyte (MK), monocyte (M) and granulocyte lineages (G). The following cytokine combinations were used: E (TPO/SCF/IL-3); G (G-CSF, GM-CSF, SCF, IL-3, IL-3); MK (TPO, SCF, IL-3) and M (M-CSF, GM-CSF, IL-6, IL-3 and SCF). Differentiation to the selected lineages was monitored every 3 days using morphology, special staining (benzidine) and flow cytometry analysis using appropriate lineage specific antibodies. Human bone marrow CD34+ and peripheral blood (PB) CD3+ (pan-T cells) and CD19+ (B-lymphocytes) selected cells were obtained from 3 different donors. Total RNA was obtained from the E and M cultures at days 7, 9, 11 and 14; G (days 10 and 14) and MK at day 14 and was hybridized in duplicate to the UCRs microarray chip (OSU version 4). After normalization of the array data with quantiles we analyzed the data using the BRB tools. Unsupervised analyses of the data revealed that samples segregated mainly in 6 main clusters corresponding to the M, E, G and Mk lineages, PB lymphocytes and CD34+ cells. Next we performed a series of two class analysis where we compared the UCRs expression of CD34+ cells vs. differentiated cells (E, G, MK, M, T and B-lymphocytes) one at a time (i.e. CD34+ cells vs. E). In table 1 we showed the top UCRs differentially expressed between in vitro differentiated HPCs/PB lymphocytes and CD34+ cells (+ or − sign reflects if the UCRs is in the sense (+) or antisense orientation (−). Table 1 Erythrocyte Megak Monocyte Granulocyte Pan T-Lym CD19+ Lym Up-regulated uc.283+ uc.285+ uc.132− uc.161+ uc.419− uc.469− uc.285+ uc.350+ uc.33+ uc.145− uc.145− uc.132− uc.10+ uc.73+ uc.420− uc.262− uc.382− uc.145− uc.188− uc.356− uc.269− uc.170− uc.170− uc.170− Down-regulated uc.43− uc.309+ uc.188− uc.160+ uc.356− uc.331+ uc.183− uc.43− uc.160+ uc.356− uc.477+ uc.183− These signatures were highly predictive since only few UCRs (mean 24) were able to predict the lineage of origin with no error (mean percentage of correct classification: 100%) after cross validation using multiples algorhytm (Class prediction within BRB). We validated the array results for uc.283+, uc.285+ and uc.161+ in a panel of differentiated CD34+ HPCs and CD34+ cells using qRT-PCR. In summary we have characterized the UCRs expression during hematopoietic differentiation of HSC and identified distinctive signatures associated with particular lineages. This research has the potential to identify novel regulators of hematopoiesis and may give insights into basic biology of gene expression and cell fate determination.

Altered microRNA (miR) expression contributes to aberrant post-transcriptional regulation of gene expression in different type of cancers; however, their role in the pathogenesis and progression of chronic myelogenous leukemia (CML) from chronic phase (CML-CP) to blast crisis (CML-BC) is still largely unknown. Microarray analysis of miR expression reveals that a discrete number of miRs are significantly upregulated (∼ 6.7% of the total 505 miRs present on the chip; 34 miRs) or downregulated (∼2.8% of the miRs present on the chip; 14 miRs) in an imatinib-sensitive manner in CML-BCCD34+ compared to CML-CPCD34+ progenitors and in BCR/ABL-expressing hematopoietic cell lines compared to untransformed parental cells. Among them, we focused our attention on miR-223, miR-15a/16-1 and miR-328, a microRNA with no currently known function, because of their importance in myelopoiesis, potential role as tumor suppressors and sequence homology with the 5’UTR of CEBPA mRNA, respectively. In 32D-BCR/ABL and K562 cells, Northern blot and TaqMan RT-PCR analyses revealed that expression of miR-223, miR-328, miR-15a and miR-16-1 was markedly suppressed (50–75% inhibition) by p210-BCR/ABL kinase activity and that imatinib treatment (1mM; 24h) restored the expression of these miRs to levels similar to those detected in non-transformed 32Dcl3 cells. Interestingly, sequence analysis of both miR-223 and miR-328 revealed homology with the hnRNP E2-binding site contained in the CEBPA uORF/spacer mRNA, a known target of the negative regulator of myeloid differentiation hnRNP E2. Accordingly, REMSA and UV-crosslinking experiments showed that synthetic miR-223 and to a greater extent miR-328 bind efficiently to recombinant hnRNP E2 protein and compete for its binding to an oligoribonucleotide containing the CEBPA uORF/spacer region, which is required for hnRNP E2-mediated translational inhibition of CEBPA in CML-BCCD34+ progenitors. Furthermore, both miR-223 and miR-328 bind endogenous hnRNP E2 from lysates of BCR/ABL-expressing but not parental cells, and from lysates of parental 32Dcl3 myeloid precursors ectopically expressing a Flag-tagged hnRNP E2 protein, suggesting that miR-223 and miR-328 may act as decoy molecules that interfere with the translation-inhibitory activity of hnRNP E2. Indeed, ectopic expression of miR-223 restored G-CSF-driven granulocytic maturation of differentiation-arrested 32D-BCR/ABL cells and restored C/EBPα expression, whereas it did not have any effect on cytokine-independent growth and clonogenic potential. Consistent with its ability to bind hnRNP E2, miR-328 also rescued C/EBPα expression and differentiation of cytokine-independent BCR/ABL-expressing myeloid precursor 32Dcl3 cells. By contrast, BCR/ABL-dependent colony formation was markedly reduced by overexpression of miR-15a and miR-16-1 (65–75% inhibition, P

MicroRNAs (miRNAs) comprise a family of small RNA, each member of which can potentially regulate post-transcription gene expression in a temporal and tissue-specific manner. Each miRNA encodes a transcript of about 22 nucleotides that can modulate expression of specific target mRNA. We identified that the chronic lymphocytic leukemia (CLL) cells of a large proportion of patients have aberrant, low-level expression of two clustered miRNAs located at 13q14, designated miR-15a and miR-16–1, providing the first example of dysregulated miRNA expression in a human cancer (PNAS USA99:15524, 2002). These miRNAs each have sequences that potentially could target mRNA encoding the anti-apoptotic protein bcl-2, which is expressed at high levels in CLL. Transfection of a lymphoma B cell line lacking expression of miR-15a and miR-16–1 with an expression vector encoding miR-15a and miR-16–1, designated pmiR-15/16, attenuated the expression-level of bcl-2 protein and enhanced the susceptibility of such lymphoma B cells to apoptosis in vitro. However, it was not certain whether miR-15a and miR-16–1 could modulate expression of bcl-2 in primary leukemia cells that were not adapted for propagation in vitro. We selected primary CLL cell samples (n = 3) that harbored deletions at 13q14 and that lacked expression of miR-15a and miR-16–1, as assessed via microRNA array and quantitative RT-PCR analyses. We transfected these cells with pmiR-15/16 or a control vector via electroporation and, in parallel studies, also transfected these CLL cells with sense and antisense control oligo-RNAs via transmessenger transfection. Before and after such manipulations we monitored for expression of miR-15a and miR-16–1 by RT-PCR, for relative expression of BCL-2-family member transcripts using a multiplex ligation-dependent probe amplification (MLPA) assay, and for expression of bcl-2 protein using immunoblot analysis and flow cytometry. We found that CLL cells transfected with either pmiR15/16 or with miR-15a and miR-16–1 sense oligo-RNAs had increased expression-levels of miR-15a and miR-16–1 within 24 hours after transfection, whereas CLL transfected with the control vector or antisense oligo-RNA did not. Depite high-level expression of miR-15a and miR-16–1, the relative levels of BCL-2 transcript did not change over the 48 hours after transfection that we examined. However, in this time period we observed that CLL cells made to express miR-15a and miR-16–1, experienced significant reductions in the levels of bcl-2 protein, which were not observed in control transfected CLL cells. This is the first demonstration that miRNAs can effect post-transcriptional regulation of protein expression in a primary human tumor and suggest that miRNAs may have potential therapeutic utility in the modulation of pathogenic gene-expression in CLL and other cancers.

We and others previously reported that leukemic CLL cells spontaneously secrete VEGF and that VEGF can enhance the leukemic B cell apoptosis resistance via unknown mechanisms. Here, we report elevated VEGF secretion is associated with CLL B cell over-expression of hypoxia inducible factor-1 alpha (HIF-1α), a transcription factor that is a key regulator of VEGF synthesis. Immunostaining analysis confirmed CLL B cells in blood and bone marrow over-expressed both HIF-1α and VEGF. The high HIF-1α levels were not linked to proline residue (402/564) mutations in oxygen dependent degradation domains; absence of proline hydroxylation dependent enzymes (PHD1, 2, 3, and FIH); or to pVHL mutation (exon 1, 2 and 3); or aberrant transcription of HIF-1α. However, pVHL, a regulator of HIF-1α degradation, was found to be lower in CLL B cells and unable to physically interact with HIF-1α in CLL B cells. Nuclear extraction assay revealed that HIF-1α interacted with its co-factor, p300/CRP in B cells, indicating that HIF-1α was transcriptionally active. We hypothesized that reduction of pVHL expression in CLL B cells could be related to repression by micro RNA function which is known to be aberrantly expressed in CLL. We focused on miR-92-1 known to be over-expressed in CLL B cells and it has pVHL as a putative target. In a luciferase assay of transfected human megakaryocytic cell line, MEG-01, we found a direct effect for miR-92-1 on pVHL, with significant repression of luciferase activity of VHL expression by miR-92-1 compared to both control vectors and a mutated target mRNA sequences. This result indicated that wild-type miR-92-1 was able to interact directly with the 3′ UTR sequence of pVHL and subsequently repress pVHL expression. To further confirm this finding, we did transient transfections of either the CLL cell line Mec-1, primary CLL B cells or normal B cells with a sense oligo miR-92-1 (both Mec-1 and CLL B cells) or wild-type miR-92-1 plasmid (normal B cells). We found the level of flow detectable pVHL lower when compared to non-transfected cells. Because we were interested in the relationship of HIF-1α to CLL B cell survival we studied if chemotherapeutic drugs can downregulate HIF-1α in CLL B cells. To do this we studied flavopiridol a drug that has very promising activity in CLL. Our results indicated that flavopiridol does down-regulate HIF-1α levels in CLL B cells. This latter change was associated with induction of apoptosis in CLL B cells. Overall, our data show that elevated VEGF secretion is related to constitutively elevated HIF-1α levels and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. We also show that elevated HIF-1α levels could be partially related to depressed pVHL with a unique mechanism for its repression: the over-expressed microRNA, miR-92-1 in CLL-B cells.

MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.

MicroRNAs are small non-coding RNAs of 19–25 nucleotides in length that are negative regulators of gene expression. Findings over the last few years indicate that microRNAs are involved in fundamental cellular process, including development and hematopoietic differentiation. Acute myeloid leukemia (AML) is a heterogeneous disorder that is characterized by proliferation of immature cells. Although there are well defined molecular subtypes of AML, the pathogenesis in the majority of cases is largely unknown. Focusing on known genes will not likely suffice to uncover the nature of the AML. The integration of a whole genome approach including non-coding RNA gene products may lead to an improve understanding of the biology of AML. Methods: To determine whether microRNAs are associated with known cytogenetic abnormalities and biological features in AML, we evaluated the microRNA expression profiles of 176 samples of adult AML with intermediate and poor risk cytogenetics and 10 CD34+ cells from healthy donors using a microarrays platform. After normalization, data were analyzed using significance analysis of microarrays and prediction analysis of microarrays software. An independent set of 28 patients with AML was used to validate the signatures using quantitative real time PCR. Treatment response was evaluated in 29 newly AML diagnosed patients 4 to 6 weeks after induction chemotherapy with idarubicin and cytarabine by bone marrow examination. Complete remission was defined as less than 5% blasts in the bone marrow. Otherwise it was categorized as resistant disease. Results: We found several microRNAs differentially expressed between CD34+ cells and all the AML samples. A subset of these microRNAs reflects the differentiation stage of the leukemias and correlate with the French-American-British classification of AML. Likewise, microRNAs are closely associated with the prevalent cytogenetic abnormalities. A common signature including the over expressed miR-20; miR-17, miR-25 and miR-191 are associated with short overall survival, while miR-29b is found down-regulated in patients with resistant disease. Furthermore, we proved experimentally that miR-29b regulates negatively MCL-1, a critical apoptosis regulator, which has been found up-regulated and associated with relapse and chemotherapy resistance in leukemia. Conclusions: MicroRNAs expression in AML is closely associated with differentiation stage, morphology and cytogenetics. A subset of MicroRNAs is correlated with survival and treatment response.

MicroRNAs (miRs) play a key role in the regulation of gene expression in normal and abnormal cell growth/differentiation, via suppression of translation or degradation of target mRNAs mainly by base pairing to their 3′ UTR sequences. However, little is known on miR expression/function in normal hematopoiesis. We have assayed the level of miRs in serum-free unilineage culture of cord blood (CB) CD34+ hematopoietic progenitor cells (HPC) through the erythroid (E), megakaryocytic (Mk), granulocytic (G) and monocytic (Mo) series at discrete stages of differentiation up to terminal maturation. The assay included control CB T cells. The analysis was performed by microarray and confirmed by Northern blot. Altogether, 49 miRs are expressed at significant levels in hematopoiesis. Cluster analysis indicates lineage-specific expression patterns. Furthermore, miR levels are mostly downmodulated through HPC differentiation/maturation. A group of 12 single or clustered miRs is sharply modulated (> 10-fold) in hematopoiesis according to specific patterns, thus suggesting their key functional role. This group includes:miR-223, downmodulated in the E series, but upmodulated in the other lineages;miR-221/222, miR-146 and miR-155 declining more sharply in E than other lineages;miR-17/20/106 and miR-25, miR-32, declining prevailingly in the G and Mo series;a miR group showing little downmodulation in the Mk or Mo lineages (miR-123, -130a or miR-181a, b, respectively);miR-99b, -100 downmodulated in all lineages;miR-210 upmodulated in all lineages. Bioinformatic analysis suggests that these miRs have important targets, which have been in part verified by the luciferase target assay (see below). Relevant examples include miR-221/222 and -130a targeting kit receptor, miR-146 targeting SDFR/CXCR4, miR 181a, b, 99b and 100, 210 targeting diverse HOX genes (respectively, HOXB5, A1 and A3). Novel miR-based regulatory circuitries have been identified:(a) miR-221/222 modulate erythropoietic and erythroleukemia cell growth via kit receptor targeting (N. Felli et al, PNAS, 2005);(b) miR-17-5p/20a/106 function as a master gene complex controlling monocytopoiesis through AML1 targeting, leading to M-CSF receptor transactivation (L. Fontana et al, this Meeting);miR-155 and -221/222 control megakaryopoiesis at progenitor and precursor level through Ets-1 multitargeting (G. Marziali et al, this Meeting).